The present invention is directed generally to a porous polymer medium, produced by photopolymerization, for separating and concentrating charged protein species from a carrier fluid that can contain both charged and uncharged species.
It is frequently the case that it is desired to analyze molecular species, and charged protein species in particular, that are present in very low concentration either because the sample itself is very small or dilute or because the species of interest is present as a consequence of prior chemical processing and is thus at very low concentration. Moreover, these molecular species can be charged and being in the presence of uncharged molecules present further difficulty in analysis or separation.
Prior art provides a plurality of methods for concentrating molecular species from solution. However, there are numerous problems associated with these prior methods, such as the need for specialized column packing, the need for specialized solvents or buffer solutions, the need to change solvents or buffer solutions in order to elute concentrated molecular species, the need to change flow direction or flow conditions between the steps of retaining and eluting species, and the inability to either separate charged and uncharged molecular species or effect an efficient separation.
Zare et al. (Anal. Chem., 73, 3921-3926, 5539-5543, 5557-5563, November 2001) describe porous sol-gel monoliths that overcome some of the problems inherent in prior art column packing materials. These monoliths, useful for capillary electrochromatography, wherein neutral species are to be separated, can be prepared by a one-step process but their pore structure is uncontrolled.
Palm et al., Anal. Chem., 69, 4499-4507, 1997, have described a one-step process for in situ preparation of macroporous polyacrylamide gel matrices for capillary electrochromatography that can be purged by the use of electroosmotic flow (EOF). While the solvent can be purged from these formulations by the use of EOF, the gel matrices have limited structural stability in useful chromatographic solvents such as acetonitrile. Moreover, polyacrylamide gels are highly swelled gels of low polymer content that rely on the solvent for their structure. Thus, these gels suffer from the draw back that they cannot be dehydrated without losing their structure.
A method for separating and concentrating charged species from uncharged or neutral species regardless of size differential has been disclosed by Singh et al. in U.S. patent application Ser. No. 09/256,586 entitled Electrokinetic Concentration of Charged Molecules, incorporated herein in its entirety. The method uses reversible electric field induced retention of charged species, that can include molecules and molecular aggregates such as dimers, polymers, multimers, colloids, micelles, and liposomes, in volumes and on surfaces of porous materials. The retained charged species are subsequently quantitatively removed from the porous material by a pressure driven flow that passes through the retention volume and is independent of direction thus, a multi-directional flow field is not required. Uncharged species pass through the system unimpeded thus effecting a complete separation of charged and uncharged species and making possible concentration factors greater than 1000-fold.
Singh et al. have found that the phenomenon of retention or trapping of charged molecules under the influence of an electric field occurs neither in an open capillary or channel nor in a capillary or channel packed with a stationary phase consisting of nonporous silica or polymer particles having a diameter of 1 xcexcm or larger. Consequently, the porous stationary phase in Singh et al. has certain required properties. First, it must be capable of supporting electroosmotic flow. Second, the porous particulate material must have certain physical characteristics in order to effectively trap and retain charged particles. Silica particles having a diameter of about 1.5 to 20 xcexcm and containing pores having a diameter of about 50 to 500 xc3x85 are preferred as a stationary phase material and silica particles having a diameter of about 5 xcexcm and containing pores having a diameter of about 300 xc3x85 are particularly preferred.
While a porous particulate stationary phase has been shown to effectively trap and retain charged particles, these stationary phases are very difficult to fabricate, particularly in microchannels. In particular, the need to retain the particulate materials within the chromatography column requires fabrication of porous frits of controlled pore size over a significant length and high mechanical stability. This requirement presents a significant challenge to the use of a porous particulate stationary phase for separation of charged from uncharged species.
Accordingly, the present invention is directed to a three-dimensional microporous polymer network material, or monolith, that can be cast-to-shape in a microchannel. The polymer monolith is fabricated with structural characteristics that provide the capability for trapping and retaining charged protein species from a mixture of charged and uncharged species under the influence of an electric field. The retained charged particles are released by application of a pressure gradient in the substantial absence of an electric field.
Application of an axial voltage differential to a solution in contact with column or microchannel containing a stationary phase, wherein the stationary phase is the porous polymer monolith of the present invention, causes the solution, that can contain both charged and uncharged protein species to move through the column under the influence of electrophoretic and/or electroosmotic forces. Neutral or uncharged species contained in the solution completely traverse the length of the column while the charged protein species, that can be smaller or bigger than the neutral species, are retained on the porous polymer monolith. Retention or trapping of charged species only occurs in the presence of a porous stationary phase and while an electric field is applied along the solution flow path.
Charged protein species retained on the porous polymer monolith are removed by application of a pressure differential to the packed column. While it is preferred that the pressure differential be applied in the substantial absence of an electric field, it has been found that by adjusting the relationship between applied pressure and voltage such that the pressure-driven flow is significantly greater than the electric field induced transport, the charged protein species can be eluted from the porous stationary phase while a voltage is being applied to the column. Further, the pressure-driven flow that removes the retained charged species from the porous matrix is independent of direction and thus neither means to reverse fluid flow nor a multi-directional flow field is required. Consequently, a single flow through the porous polymer monolith can be employed to separate charged from uncharged protein species in contrast to prior art systems.
The polymer monolith is produced by a phase separation process. Phase separation occurs during polymerization of a monomer or monomer mixture dissolved in a solvent yielding a polymer-rich and a solvent-rich phase. The solvent-rich phase produces the micropores through which liquids flow during a separation. The parameters of the phase separation process are established such that the solvent-rich phase forms a majority of pores in the range of about 200 nm-10 xcexcm. The lower limit (≈200 nm) is established to provide efficient hydrodynamic flow of a liquid phase during either pressure-driven or electric field-driven flow. The upper limit (≈10 xcexcm) must be small enough for interaction by diffusion between mobile and stationary phases. The polymer-rich phase becomes a porous monolithic backbone that is formulated to contain a substantial portion of an alkoxy-substituted metal, such as Si, Ti, or Zr, as a protected functionality. Hydrolysis of the alkoxy functionality provides the charged groups necessary for electric field-driven flow. Consequently, at least about 20 vol % of the monomers contain an alkoxy protected metal functionality.
Extensive crosslinking allows the porous polymer monoliths to achieve high molecular weights and, in contrast to prior art porous polymer materials, imparts a high structural stability such that the polymer monolith resists swelling and/or dissolution in the presence of a wide variety of solvents and during subsequent hydrolysis. In the present invention at least about 20 vol % of the monomer is present as a crosslinker in order to provide a rigid matrix such that hydrolysis of the alkoxymetal functionality will not collapse the structure. If the monolith is to be employed as a separations medium in reverse phase chromatography applications, it is desirable to have at least about 25 vol % of the monomer containing a hydrophobic group such as alkyl, aryl, or substituted versions thereof.
Polymerization is initiated by adding a polymerization initiator and exposing the solvent/monomer solution to radiation whose wavelength is matched to the absorbance of the free radical polymerization initiator. Following the phase separation step and after the three-dimensional polymer structure has been established, the alkoxy protecting groups are hydrolyzed. This converts a portion of the monomer to a porous metal oxide structure that cannot collapse despite the large localized loss of mass because the bulk structure of the monolith has been defined by the initial crosslinked vinyl polymerization step. In this way, the polymer of the invention differentiates from sol/gel produced polymer materials. The charge on the metal oxide, and consequently the zeta potential, is controlled by pH, i.e., silica is negative at pHxe2x89xa74 and titania is positive at pHxe2x89xa66. After hydrolysis, the solution remaining in the pores of the monolith is exchanged for an appropriate running buffer.
In addition to separating charged from uncharged protein species, the monolithic polymer material produced by the invention has been found to function as a chromatographic medium. The inventors have found that when a plurality of retained charged protein analytes are eluted from the polymer stationary phase by application of a pressure gradient, a chromatographic peak relating to each of the plurality may be observed.